Historically, the present state of knowledge regarding DNA polymerases as a broad category of enzymes stems from the in-vitro synthesis of DNA first accomplished by Arthur Kornberg and his co-workers who in 1957 isolated an enzyme from E. coli bacteria now known as DNA polymerase I. This enzyme was found to catalyze the covalent addition of nucleotides to pre-existing DNA chains. Since the initial discovery of DNA polymerase I, a large number of different DNA polymerases have been isolated and characterized from different bacteria, viruses, and animal cells. Three different DNA polymerases (I, II, and III) have been identified and extensively studied in E. coli and B. subtilius bacterial strains. Investigations of many retroviruses has revealed the presence of RNA directed DNA polymerases, commonly termed "reverse transcriptases", which are presently thought to play a central role in the viral propagation of acquired immunodeficiency syndrome and feline leukemia. Such reverse transcriptases have been isolated from retrovirally infected cells of animals and humans as well.
Distinct from these are the three major DNA polymerases (alpha, beta, and gamma) which have been isolated from and identified in a variety of different eukaryotic cells. In eukaryotes, it is DNA polymerase alpha which is responsible for the replication of chromosomal DNA while polymerases beta and gamma synthesize other forms of DNA. More recently, a fourth DNA polymerase, termed delta, has been isolated from calf thymus and rabbit bone marrow but its significance is not yet known. In addition to these DNA polymerases in eukaryotes, there also exists terminal deoxynucleotidyl transferase whose presence is limited to primitive lymphocytes and to certain types of leukemia and lymphoma cells; and whose physiological function remains unknown. Clearly, there is a wide variety and diversity of enzymes which are recognized, characterized, and grouped under the broad category of DNA polymerases.
With the recognition of the variety and diversity of individual enzymes within the broad DNA polymerase category, the approach of selectively inhibiting individual DNA polymerases has been recognized and exploited not only for a definition of individual DNA polymerase function and activity but also as an effective means for therapeutically treating viral infections and neoplastic diseases and disorders [Plunkett and Cohen, Cancer Research 35:1547-1554 (1975); Major et al., Biochemical Pharmacology 31:2397 (1982); Kufe et al., J. Biol. Chem. 255:8997 (1980); Mitsuya and Broder, Nature 325:773-778 (1987)]. For some years, interest has been focused on the use of 2',3'-dideoxynucleotides such as 2',3'-dideoxyadenosine triphosphate (hereinafter "ddATP") and 2',3'-dideoxythymidine triphosphate (hereinafter "ddTTP") as compositions which differentially inhibit a variety of different eukaryotic and viral DNA polymerases [Toji and Cohen, Proc. Natl. Acad. Sci. USA 63:871-877 (1969); Toji and Cohen, J. Bacteriol. 103:323-328 (1970); Edenberg et al., J. Biol. Chem. 253:3273-3280 (1978); Allaudeen, H.S., Biochem. Pharmacol. 29:1149-1153 (1980); Krokan et al., Biochemistry 18:4431-4443 (1979); and Ono et al., Biochem. Biophys. Res. Comm. 88:1255-1262 (1979)]. Structurally, the 2',3'-dideoxynucleosides (hereinafter "ddNs") are analogues of the normal 2'-deoxynucleosides (hereinafter "dNs") which lack the 3'-OH group of the ribose sugar moiety. If the ddNs are phosphorylated to 2', 3'-deoxynucleoside-5'-triphosphates (hereinafter "ddNTPs") or dideoxynucleotides, they become analogues of the 2'-deoxynucleoside-5'-triphosphates (hereinafter "dNTPs") which normally serve as substrates for cellular and viral DNA polymerases. Generally, however, it is agreed by investigators in this art that the rationale and the mechanism(s) of action by which specific DNA polymerases do or do not utilize one or more ddNTPs as substrate analogues is not clear or understood; and that there is no predictive value or expectation that specific dideoxynucleoside or dideoxynucleotide would be recognized by any individual DNA polymerase [Wagar et al., J. Cell. Physiol. 121:4022-408 (1984) and the references cited therein].
This lack of predictive expectations is well demonstrated by the recent intensity and focus on the use of 2', 3'-dideoxynucleotides as anti-retroviral agents, particularly with regard to HIV (HTLV-III/LAV) infections and acquired immunodeficiency syndrome or AIDS. The retroviral reverse transcriptases, the viral DNA polymerases, are able to utilize the triphosphorylated dideoxynucleosides (ddNTPs) as substrate analogues--the consequence of which is that further nucleotide addition becomes impossible due to the absence of a 3'-OH group on the ribose moiety. Through this technique of chain termination, proviral DNA synthesis is prevented and retroviral proliferation is inhibited [Mitsuya and Broder, Nature 325:773-778 (1987); Balzarini et al., Biochem. Biophys. Res. Comm. 140:735-742 (1986); Mitsuya and Broder, Proc. Natl. Acad. Sci. USA 83:1911 (1986); Mitsuya et al., Proc. Natl. Acad. Sci. USA 82:7096 (1985); Smoler et al., J. Biol. Chem. 246: 7697 (1971); Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977)].
In comparison to other members of the DNA polymerase category, terminal deoxynucleotidyl transferase (hereinafter "TdT") has remained a relatively unknown and uncharacterized enzyme. Although the existence of TdT has been known for twenty-five years, its physiological function in the cells in which it is expressed, whether normal or malignant, remains unknown. Terminal deoxynucleotidyl transferase, however, has been demonstrated to be a unique DNA polymerase which catalyzes the polymerization of deoxyribonucleotides on the 3'-hydroxyl ends of preformed oligo- or polydeoxynucleotide initiators, in a template independent manner [Bollum, F.J., in The Enzymes: Terminal Deoxynucleotidyl Transferase (R.D. Boyer, editor), Academic Press Inc., New York, 1974, p 145]. Its expression is restricted, in normal animals, to subsets of primitive lymphocytes and, in disease states, to the blast cells of certain forms of acute leukemia and diffuse lymphoma [McCaffrey et al., Cancer Research 41:4814 (1981)]. For immunobiologists, TdT has emerged as a useful marker for characterizing subsets of pre-B lymphocytes and pre-T lymphocytes [Silverstone et al., J. Exp. Med. 144:453 (1976); Janossy et al., J. Immunol. 123:1525 (1979); Bollum, F.J., Blood 54:1203 (1979); Blatt et al., NEJM 303:918 ( 1980); Greaves, M.F., Cancer Research 41:4752 (1981)]. For physicians clinically treating leukemias and lymphomas, neoplastic cell TdT status has become a useful criterion for patient assignment to therapeutically meaningful categories [Marks et al., NEJM 298:812 (1978)].
Due to the presence of terminal deoxynucleotidyl transferase in some leukemias and lymphomas, a strategy for the treatment of such diseases using specific inhibitors of TdT has been suggested and empirically attempted. To date, such investigations have focused on the use of 6-anilinouracil derivatives as both specific compositions and selective methods for inhibiting the activity of terminal deoxyribonucleotidyl transferase [McCaffrey et al., in New Experimental Modalities In The Control Of Neoplasia (P. Chandra, editor), NATO ASI Series A, Life Sciences Volume 120, 1986, page 213; U.S. Pat. No. 4,576,948]. These studies have established that TdT is important for the viability of the cells in which it is expressed: the inhibition of TdT by 6-anilinouracils results in cytotoxic damage to TdT-positive cells. However, 6-anilinouracils are not readily soluble except in dimethylsulfoxide, a circumstance which inhibits their clinical utility. Moreover, in light of the dismal prognosis for patients afflicted with TdT-positive diseases (typically, only several months), there remains a continuing need for therapeutically effective compositions in the treatment of human leukemias and lymphomas using TdT as a therapeutic target. Accordingly, the development of additional therapeutic compositions which would selectively eliminate TdT-positive neoplastic cells would be recognized as a major advance and welcome improvement upon the presently available means for therapeutic treatment for TdT-positive leukemias and lymphomas.